MOLECULAR MECHANISMS OF MECHANOSENSITIVE CHANNEL GATING

机械敏感通道门控的分子机制

• 批准号: 6086569
• 负责人: PAUL BLOUNT
• 金额: $ 27.14万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-06-01 至 2005-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6086569
• 关键词: bacterial proteins; biological signal transduction; cysteine; electrophysiology; intermolecular interaction; membrane channels; molecular genetics; site directed mutagenesis; structural biology; voltage /patch clamp; voltage gated channel

Bacterial mechanosensitive channels are emerging as molecular paradigms for investigation of the mechanosensory transduction that occurs in physiological processes such as touch, proprioception, cardiovascular regulation, hearing and balance. The use of the MscL channel of Escherichia coli has advanced the field considerably by allowing molecular genetic analysis to be combined with electrophysiology and transport assays in the study of a protein with a well-defined physiological role. Hence, MscL serves as a good model for determining the molecular mechanisms of mechanosensitive channel gating as well as the general principles of how proteins detect and respond to membrane tension. Purification of the MscL channel protein led to the identification of the structural gene and ultimately to a 3.5 angstrom X-ray crystallographic structure of the Mycobacterium tuberculosis MscL. This is a closed structure, which unfortunately provides few clues to the process of channel gating or to the structure of the open channel. Because a large pore of 30 to 40 angstrom diameter is generated upon gating, a large conformational change must occur and several residues normally embedded in protein or lipid environments must contribute to the lining of the open pore. The residue interations that are strongest and of greatest importance for keeping the channel closed, the movements of residues and domains that take place upon gating, and the residues that contribute to the lining of the open pore are currently all unknown. The experiments in this application are designed to use molecular analyses based on the solved structure to determine these functional and structural properties of the channel. The approaches include: 1) Cysteine scanning to determine which interactions within the transmembrane domains are of most importance in MS channel gating. 2) Utilizing the "Substituted Cysteine Accessibility Method" (SCAM) to identify the residues that move into an aqueous environment upon channel opening, and thus are likely candidates for lining the open-channel pore. 3) Isolating and characterizing suppressor mutations of gain-of-function MscL mutants to identify pairs of residues that potentially interact in the closed, transition or open states.

细菌机械敏感通道正在以分子形式出现 研究发生在大脑中的机械感觉转导的范例 生理过程，如触摸、本体感觉、心血管 调节、听觉和平衡。大肠杆菌MscL通道的应用 大肠杆菌通过允许分子遗传分析， 与电生理学和转运试验相结合， 具有明确生理作用的蛋白质。因此，MscL作为一个很好的 一个确定机械敏感通道分子机制的模型 门控以及蛋白质如何检测和响应的一般原则 膜张力MscL通道蛋白的纯化导致了 鉴定结构基因，并最终进行3.5埃的X射线检查 结核分枝杆菌MscL的晶体结构。这是一 封闭的结构，不幸的是，它提供了一些线索的过程， 通道门控或开放通道的结构。因为一个大毛孔 在门控时产生30至40埃的直径， 必须发生变化，通常嵌入蛋白质或脂质中的几个残基 环境必须有助于开放孔隙的衬里。将残余物 最强的和最重要的相互作用，以保持 通道关闭，残基和域的运动发生在 门控，和残留物，有助于衬里的开放孔是 目前都是未知数。此应用程序中的实验旨在使用 分子分析的基础上解决的结构，以确定这些功能 以及通道的结构特性。这些方法包括：1）半胱氨酸 扫描以确定跨膜结构域内的哪些相互作用 这在MS信道选通中是最重要的。2)利用“取代的半胱氨酸” 可及性方法”（SCAM），以确定移动到水溶液中的残留物 在通道开放时的环境，因此可能是衬 明渠孔隙3)分离和表征的抑制突变 功能获得性MscL突变体，以鉴定可能 在封闭、过渡或开放状态下相互作用。